Acyl Carrier Proteins (ACPs) play important roles in a number of biosynthetic pathways that are dependent upon acyl group transfers [1]. They are most often associated with the biosynthesis of fatty acids [2,3], but they are also utilized in the synthesis of polyketide antibiotics [4,5], non-ribosomal peptides [6,7], and of intermediates used in the synthesis of vitamins such as the protein-bound coenzymes, lipoic acid [8] and biotin [9]. The ACP in each of these pathways is composed of 80–100 residues and is either an integrated domain in a larger multi-functional protein (Type I synthase complex) or is a structurally independent protein that is part of a non-aggregated multi-enzyme system (Type II synthase complex). Type I synthases are found in mammals, fungi and certain Mycobacteria while type II ACPs are utilized by plants and most bacteria. The Escherichia coli ACP for fatty acid synthesis has been over-expressed [10] and purified [11,12], and the solution structure has been solved by NMR spectroscopy [13]. The fact that these proteins are essential for the maturation of the organism has led to their investigation as targets for the development of new anti-microbial agents [14–18].
ACPs require post-translational modification for activity. They are converted from an inactive apo-form to an active holo-form by the transfer of the 4′-phosphopantetheinyl (P-pant) moiety of coenzyme A to a conserved serine on the ACP. The β-hydroxy side chain of the serine residue serves as a nucleophilic group attacking the pyrophosphate linkage of CoA. Evidence now suggests [19] that each synthase that is dependent upon P-pant attachment for activation has its own partner enzyme responsible for this attachment.
The post-translational modification of the ACP subunit in the fatty acid synthase is performed by holo-[acyl carrier protein] synthase (hereinafter defined as “ACPS”; Enzyme Commission No. 2.7.8.7). The best characterized member of the ACPS family is the E. coli ACPS [20]. The enzyme produces holo-ACP by transferring the P-pant moiety to Ser-36 of the E. coli apo-ACP (SEQ ID NO:15) in a magnesium dependent reaction [20] as follows:

The over-expression and purification of the E. coli ACPS has been described [21] and this protein is classified as a member of a new enzyme superfamily, the phosphopantetheinyl transferases [19]. Based on the size of the proteins, the P-pant transferase superfamily can be roughly divided into two subgroups [22]. Enzymes responsible for modifying the peptidyl carrier protein subunits of non-ribosomal peptide synthetases are good examples for the first subgroup, which are usually ˜230 amino acids in size. The structure of one of this subgroup enzymes, the surfactin synthetase activating enzyme Sfp, has been solved recently and it consists of a 2-fold intramolecular pseudosymmetry with the CoA binding site at the interface of the symmetrical fold [22]. ACPS and other enzymes transfering the P-pant group onto domains of the fatty acid synthases are usually smaller, about ˜120 residues, and belong to the second subgroup of the P-pant transferase superfamily. The sequence homology between these two subgroups is rather low, about 12–22% between E. coli ACPS and B. subtilis Sfp, for example, although both have been shown to possess P-pant transferase activity. Alignment [19] of some of these proteins show that two regions, residues 5–13 and 54–65 (E. coli ACPS numbering), are highly conserved with five of the residues in these regions identical.
While numerous members of the phosphopantetheinyl transferase superfamily have been identified and sequenced, until the present invention, the crystal structure of ACPS complexed with halo-ACP, and the three dimensional structure of the ACPS/ACP active site has not been determined. Further, prior to the present invention, the solution structure of B. subtilis ACP had not been determined.